The objectives of the proposed research project are to obtain a better understanding of the mechanisms of several active transport systems and to use the methods developed in these studies to pursue the more general question of membrane structure and function, particularly the involvement of proteins in transport and membrane structure. A number of so called binding proteins have recently been isolated from bacterial surfaces. These proteins appear to be involved in the transport of the various small molecules which they bind. From the growing list of binding proteins five were chosen for these studies. These include the following binding proteins; leucine-isoleucine-valine, leucine-specific, isoleucine-preferring, ribose, glutamate-aspartate and cystine. These proteins may be purified to homogeneity in good yield and crystallized in only three or four steps. It is therefore feasible to prepare radioactive binding proteins which may be used to directly measure the interaction of a binding protein with a cell membrane or a membrane vesicle. In addition to providing a means of optimizing the conditions for the reassociation of binding proteins with membranes, the use of radioactive membrane proteins provides an approach to the more general question of the factors involved in protein membrane interactions. In addition to the use of radioactive binding proteins, conventional genetic techniques will be used in the characterization of the transport systems. Antibodies prepared against the binding proteins will be used in attempts to find the cellular localization of the binding proteins as well as in the studies of the function of the binding proteins. BIBLIOGRAPHIC REFERENCES: Oshima, R. G., Willis, R. C., Furlong, C. E., and Schneider, J. A. (1975). Binding assays for amino acids: The utilization of a cystine binding protein from Escherichia coli for the determination of acid soluble cystine in small physiological samples. J. Biol. Chem. 249, 6033,6039. Willis, R. C. and Furlong, C. E. (1975) Purification and properties of a ribose-binding protein from Escherichia coli. J. Biol. Chem. 249, 6926-6929.